JP2006340603A - Method for producing l-glutamic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new technology for improving L-glutamic acid productivity in the production of L-glutamic acid using coryneform bacteria. <P>SOLUTION: The method for producing L-glutamic acid comprises culturing coryneform bacteria capable of L-glutamic acid production in a culture medium so as to attain formation and accumulation of L-glutamic acid in a culture product and collecting L-glutamic acid from the culture product, wherein use is made of coryneform bacteria modified so as to increase the activity of the following protein: (A) protein having a specific amino acid sequence or (B) protein composed of the specific amino acid sequence having undergone substitution, deletion, insertion or addition of one or some amino acids, in which the protein when introduced in coryneform bacteria, exhibits the activity of enhancing the potency for L-glutamic acid production. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

【００６５】
＜４＞野生型ORF1554を搭載したプラスミドの作製
配列番号１の塩基配列を参考に、配列番号９及び１０に示すプライマーを設計し、ブレビバクテリウム・ラクトファーメンタムATCC13869から、野生型ORF1554の上流約750bpと下流約30bpを含む領域を、PCRにより取得した。PCRは、pyrobest DNA polymerase（宝酒造(株)）を用い、94℃ 5分の後、98℃ 5秒、65℃ 10秒、72℃ 60秒を30サイクル行った。取得した約1.5kbのPCR産物を、両プライマー上に設計しておいたXbaIサイトで切断し、プラスミドベクターpSAC4のXbaIサイトに挿入し、pD5WT-1を作製した。
【００６６】
＜５＞ORF1554、ORF1554^*遺伝子増幅株によるＬ−グルタミン酸生産
ORF1554、ORF1554^*について、遺伝子増幅によるＬ−グルタミン酸生産への効果を調べるため、プラスミドpD5WT-1（ORF1554）及びpD5-2A（ORF1554^*）をブレビバクテリウム・ラクトファーメンタムATCC13869由来のsucA遺伝子欠損株L30-2株に導入し、ORF1554の増幅株L30-2/pD5WT-1株、及びORF1554^*の増幅株L30-2/pD5-2A株を作製した。プラスミドの導入は電気パルス法（特開平2-207791号公報）によって行った。なお、L30-2株は、ブレビバクテリウム・ラクトファーメンタムATCC13869のsucA遺伝子欠損株ΔＳ株（WO95/34672号国際公開パンフレット）から、CM2Bプレート（10g/Lポリペプトン、10g/Lイーストエキストラクト、5g/L NaCl、10μg/Lビオチン、20g/L寒天、pH7.0）上で良好なコロニー形成をする株として単離した。
【００６７】
取得したORF1554、ORF1554^*増幅株をCMDXプレート（5g/Lグルコース、10g/Lペプトン、10g/Lイーストエキストラクト、1g/L KH₂PO₄、0.4g/L MgSO₄・7H₂O、10mg/L FeSO₄・7H₂O、10mg/L MnSO₄・4-5H₂O、3g/L尿素、2g/L（N量として）「豆濃」、20g/L寒天、5μg/Lクロラムフェニコール、pH7.5）で30℃、一晩培養後、1/6プレート分の菌体を20mlの培地（30g/Lグルコース、15g/L (NH₄)₂SO₄、0.4g/L MgSO₄・7H₂O、1mg/L FeSO₄・7H₂O、1mg/L MnSO₄・4-5H₂O、200μg/LビタミンB1、200μg/Lビオチン、0.48g/L（N量として）「豆濃」、1g/フラスコ CaCO₃、5μg/Lクロラムフェニコール、pH8.0）の入ったフラスコに接種し、30℃、20時間の振とう培養の後、バイオテックアナライザー（サクラ精機）を使用して、培地中のグルコース及びＬ−グルタミン酸の濃度を測定した。図１に、消費グルコースに対するＬ−グルタミン酸の収率と最終OD（620nm、培養液を51倍に希釈して測定）を示した。ORF1554及びORF1554^*の増幅は、いずれもブレビバクテリウム・ラクトファーメンタムのグルタミン酸収率を約4%向上させることがわかった。
【００６８】
【発明の効果】
本発明により、ブレビバクテリウム・ラクトファーメンタムのＬ−グルタミン酸生産能を向上させることができる。
【００６９】
【配列表】

【図面の簡単な説明】
【図１】 ORF1554及びORF1554^*を増幅したブレビバクテリウム・ラクトファーメンタムsucA欠損株の生育とＬ−グルタミン酸収率を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the fermentation industry, and in particular, to a method for producing L-glutamic acid and a bacterium used therefor. L-glutamic acid is widely used as a seasoning material.
[0002]
[Prior art]
Conventionally, L-glutamic acid is industrially produced by fermentation using coryneform bacteria belonging to the genus Brevibacterium or Corynebacterium having the ability to produce L-glutamic acid. In order to improve the productivity of these coryneform bacteria, strains isolated from nature or artificial mutants of the strains are used.
[0003]
In addition, various techniques for increasing L-glutamic acid-producing ability by enhancing L-glutamic acid biosynthetic enzymes by recombinant DNA techniques have been disclosed.
Furthermore, a method has been developed in which fermentation is performed while precipitating L-glutamic acid, which accumulates microorganisms having L-glutamic acid-producing ability in the culture medium under acidic conditions. Conventionally, since normal L-glutamic acid-producing bacteria cannot grow under acidic conditions, L-glutamic acid fermentation has been carried out under neutral conditions, but Enterobacter found as a microorganism having L-glutamic acid-producing ability under acidic conditions. -Agromerans can be produced and accumulated while precipitating L-glutamic acid in the medium by culturing it in a liquid medium whose pH is adjusted to a condition where L-glutamic acid is precipitated (Patent Document 1).
[0004]
By the way, the whole genome sequence of Corynebacterium glutamicum ATCC13032 has been determined and published (Non-patent Document 1). A putative ORF having high homology with ORF1554 is known (Non-patent Document 2), but its function is not known.
[0005]
[Patent Document 1]
European Patent Application No. 1078989
[Non-Patent Document 1]
DDBJ / EMBL / GenBank accession # BA000036
[Non-Patent Document 2]
DDBJ / EMBL / GenBank accession # AP005277-302
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel technique for improving L-glutamic acid productivity in the production of L-glutamic acid using coryneform bacteria.
[0007]
[Means for Solving the Problems]
In the course of conducting research on genes involved in acid resistance of coryneform bacteria, the present inventors increased the activity of a gene product of unknown function named ORF1554 among them, thereby causing L- The present inventors have found that the ability to produce glutamic acid can be improved and have completed the present invention.
That is, the present invention is as follows.
[0008]
(1) In a method for producing L-glutamic acid, a coryneform bacterium having L-glutamic acid-producing ability is cultured in a medium, L-glutamic acid is produced and accumulated in the culture, and L-glutamic acid is collected from the culture. The bacterium is modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) an activity comprising the amino acid sequence of SEQ ID NO: 2 comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and improving the ability to produce L-glutamic acid of coryneform bacteria A protein having
(2) The method according to (1), wherein the protein is encoded by the DNA shown in (a) or (b) below.
(A) DNA having a base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1.
(B) DNA encoding a protein that hybridizes with the base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1 under the stringent conditions and has an activity of improving the ability to produce L-glutamic acid of coryneform bacteria.
(3) The bacterium increases the copy number of the gene encoding the protein shown in (A) or (B), or the gene encoding the protein shown in (A) or (B) in the bacterial cell The method according to (1) or (2), wherein the activity of the protein in the cell is increased by modifying an expression regulatory sequence of the same gene so that expression of the gene is enhanced.
(4) The method according to any one of (1) to (3), wherein the protein has an amino acid sequence in which the glutamic acid residue at position 81 of SEQ ID NO: 2 is substituted with a glycine residue.
(5) The method according to any one of (1) to (4), wherein the bacterium is deficient in α-ketoglutarate dehydrogenase.
(6) A coryneform bacterium that has an ability to produce L-glutamic acid and has been modified so that the activity of the protein shown in (A) or (B) below is increased.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) an activity comprising the amino acid sequence of SEQ ID NO: 2 comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and improving the ability to produce L-glutamic acid of coryneform bacteria A protein having
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0010]
<1> Coryneform bacterium of the present invention
The coryneform bacterium of the present invention is a coryneform bacterium that has L-glutamic acid-producing ability and has been modified so that the activity of the protein shown in (A) or (B) below is increased.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) an activity comprising the amino acid sequence of SEQ ID NO: 2 comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and improving the ability to produce L-glutamic acid of coryneform bacteria A protein having
[0011]
Hereinafter, the protein (A) or (B) may be referred to as “ORF1554 product”, and the DNA encoding the protein may be referred to as “ORF1554”. In addition to ORF1554, an expression regulatory sequence such as a promoter adjacent to the ORF may be referred to as “ORF1554” for convenience.
[0012]
In the present invention, the “coryneform bacterium” has been conventionally classified into the genus Brevibacterium, but includes bacteria that are currently classified into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 ( 1981)), and Brevibacterium spp. Closely related to Corynebacterium spp. Examples of such coryneform bacteria include the following.
[0013]
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium carnae
Corynebacterium glutamicum
Corynebacterium Lilium
Corynebacterium melacecola
Corynebacterium thermoaminogenes
Corynebacterium herculis
Brevibacterium divaricatam
Brevibacterium flavum
Brevibacterium immariophilum
Brevibacterium lactofermentum
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Corynebacterium ammoniagenes
Brevibacterium album
Brevibacterium cerinum
Microbacterium / Ammonia Philus
Specifically, the following strains can be exemplified.
[0014]
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia philus ATCC15354
[0015]
You can get them from, for example, the American Type Culture Collection. That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. In addition, AJ12340 shares were issued on October 27, 1987 at the Institute of Biotechnology, National Institute of Technology, Ministry of International Trade and Industry (currently the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center) (Ibaraki, 305-5466, Japan) It is deposited under the Budapest Treaty under the accession number of FERM BP-1539 in 1st, 1st East, 1st Street, Tsukuba City. In addition, AJ12418 stock was deposited with the reference number FERM BP-2205 to the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on January 5, 1989, based on the Budapest Treaty.
[0016]
In the present invention, “L-glutamine producing ability” refers to the ability to accumulate L-glutamine in the medium when the coryneform bacterium of the present invention is cultured. This ability to produce L-glutamine may be a property of a wild strain of coryneform bacteria, or may be a property imparted or enhanced by breeding.
[0017]
To impart or enhance L-glutamine-producing ability by breeding, impart 6-diazo-5-oxo-norleucine resistance (Japanese Patent Laid-Open No. 3-232497), impart purine analog resistance and / or methionine sulfoxide resistance A method for imparting resistance to α-ketomalonic acid (Japanese Patent Laid-Open No. 56-151495), a method for imparting resistance to a peptide containing glutamic acid (Japanese Patent Laid-Open No. 2-186994), and the like. It is done.
[0018]
Specific examples of coryneform bacteria having the ability to produce L-glutamine include the following strains.
Brevibacterium flavum AJ11573 (FERM P-5492) See JP 56-151495 A
Brevibacterium flavum AJ12210 (FERM P-8123) See JP-A-61-202694
Brevibacterium flavum AJ12212 (FERM P-8123) See JP-A-61-202694
Brevibacterium flavum AJ12418 (FERM-BP2205) See JP-A-2-186994
Brevibacterium flavum DH18 (FERM P-11116) See JP-A-3-232497
Corynebacterium melacecola DH344 (FERM P-11117) Refer to Japanese Patent Laid-Open No. 3-232497
Corynebacterium glutamicum AJ11574 (FERM P-5493) See JP-A-56-151495
[0019]
“Modified to increase the activity of the ORF1554 product in the cell” means that the activity per cell is higher than that of an unmodified strain such as a wild-type coryneform bacterium. For example, the case where the number of ORF1554 product molecules per cell increases or the activity per ORF1554 product molecule increases. The wild-type coryneform bacterium to be compared is, for example, Brevibacterium lactofermentum ATCC13869. When the activity of the ORF1554 product of the coryneform bacterium is increased, the ability of the coryneform bacterium to produce L-glutamic acid is improved. In the present invention, “activity for improving L-glutamic acid-producing ability of coryneform bacteria” refers to the activity of such an ORF1554 product. Specifically, when a strain of coryneform bacterium modified so as to express the ORF1554 product in excess over the wild strain or the non-modified strain is cultured in the medium, L-glutamic acid is more effective than the wild strain or the non-modified strain. If the amount of accumulation in the medium is large or the production rate of L-glutamic acid is high, it can be said that the modified strain has improved L-glutamic acid production ability.
[0020]
Enhancement of ORF1554 product activity in coryneform bacterial cells is achieved by enhancing ORF1554 expression. Enhancement of the expression level of the gene is achieved by increasing the copy number of ORF1554. For example, an ORF1554 fragment may be ligated with a vector that functions in the bacterium, preferably a multicopy type vector, to produce a recombinant DNA, which is introduced into a host capable of producing L-glutamine and transformed. . Alternatively, the recombinant DNA may be introduced into a wild-type coryneform bacterium to obtain a transformed strain, and then the L-glutamine-producing ability may be imparted to the transformed strain.
[0021]
As ORF1554, any of genes derived from coryneform bacteria and genes derived from other organisms such as bacteria belonging to the genus Escherichia can be used. Of these, genes derived from coryneform bacteria are preferred from the viewpoint of ease of expression.
[0022]
The sequence of Brevibacterium lactofermentum ORF1554 has been clarified by the present invention (SEQ ID NO: 1), and the gene presumed to be a homologue of ORF1554 of Corynebacterium glutamicum has already been sequenced. (DDBJ / EMBL / GenBank accession # AP005277-302), PCR using the chromosomal DNA of coryneform bacteria as a template using primers prepared based on these nucleotide sequences, for example, the primers shown in SEQ ID NOs: 9 and 10 ORF1554 and its adjacent region can be obtained by the method (PCR: polymerase chain reaction; see White, TJ et al., Trends Genet. 5, 185 (1989)). Homologs of ORF1554 of other microorganisms can be obtained in the same manner.
[0023]
Chromosomal DNA is obtained from bacteria that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Nippon Biotechnology, Ed., Pp. 97-98, Bafukan, 1992).
[0024]
ORF1554 amplified by PCR is prepared by connecting recombinant DNA into autonomously replicating vector DNA in cells of Escherichia coli and / or coryneform bacteria, and introducing this into Escherichia coli. This makes it easier to operate later. Examples of vectors capable of autonomous replication in Escherichia coli cells include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, and pMW219.
[0025]
The vector that functions in coryneform bacteria is, for example, a plasmid that can autonomously replicate in coryneform bacteria. Specific examples include the following.
pAM330 See JP-A-58-67699
pHM1519 See JP-A-58-77895
[0026]
In addition, DNA fragments capable of autonomously replicating plasmids in coryneform bacteria can be taken out of these vectors and inserted into the Escherichia coli vector, allowing autonomous replication in both Escherichia coli and coryneform bacteria. It can be used as a so-called shuttle vector.
[0027]
Examples of such a shuttle vector include the following. The microorganisms holding each vector and the accession number of the international depositary organization are shown in parentheses.
pAJ655 Escherichia coli AJ11882 (FERM BP-136)
Corynebacterium glutamicum SR8201 (ATCC39135)
pAJ1844 Escherichia coli AJ11883 (FERM BP-137)
Corynebacterium glutamicum SR8202 (ATCC39136)
pAJ611 Escherichia coli AJ11884 (FERM BP-138)
pAJ3148 Corynebacterium glutamicum SR8203 (ATCC39137)
pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)
pHC4 Escherichia coli AJ12617 (FERM BP-3532)
[0028]
These vectors are obtained from the deposited microorganism as follows. Cells collected in the logarithmic growth phase are lysed using lysozyme and SDS, centrifuged at 30000 × g, polyethylene glycol is added to the supernatant obtained from the lysate, and cesium chloride-ethidium bromide equilibrium density gradient centrifugation. Separate and purify by separation.
[0029]
In addition, plasmids pSFK6 (Japanese Patent Laid-Open No. 2000-262288, US Pat. No. 6,303,383) and pSAC4 (US Pat. No. 5,846,790) shown in Examples below can also be used as shuttle vectors.
[0030]
To prepare a recombinant DNA by linking ORF1554 and a vector that functions in coryneform bacteria, the vector is cleaved with a restriction enzyme that matches the end of ORF1554. Ligation is usually performed using a ligase such as T4 DNA ligase.
[0031]
In order to introduce the recombinant DNA prepared as described above into a coryneform bacterium, transformation methods that have been reported so far may be used. For example, as reported for Escherichia coli K-12, a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. , 53, 159 (1970)), and a method for introducing competent cells from cells at the growth stage and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE). Gene, 1, 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily incorporates recombinant DNA. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A. Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). In addition, transformation of coryneform bacteria can also be performed by the electric pulse method (Japanese Patent Laid-Open No. 2-207791).
[0032]
Increasing the copy number of ORF1554 can also be achieved by having multiple copies of ORF1554 on the chromosomal DNA of coryneform bacteria. In order to introduce ORF1554 in multiple copies on the chromosomal DNA of coryneform bacteria, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in JP-A-2-109985, ORF1554 can be mounted on a transposon, transferred, and introduced in multiple copies onto chromosomal DNA.
[0033]
In addition to the gene amplification described above, the enhancement of ORF1554 product activity can also be achieved by replacing an expression regulatory sequence such as a promoter of ORF1554 on a chromosomal DNA or plasmid with a strong one. For example, lac promoter, trp promoter, trc promoter and the like are known as strong promoters. Further, as disclosed in International Publication WO00 / 18935, it is possible to introduce a base substitution of several bases into the promoter region of ORF1554 and modify it to be more powerful. These promoter substitutions or modifications enhance ORF1554 expression and enhance ORF1554 product activity. Modification of these expression control sequences may be combined with increasing the copy number of ORF1554.
[0034]
The replacement of the expression regulatory sequence can be performed, for example, in the same manner as the gene replacement using a temperature sensitive plasmid. Examples of temperature sensitive plasmids of coryneform acid bacteria include p48K and pSFKT2 (see JP 2000-262288 A), pHSC4 (French Patent Publication 1992 2667875, JP 5-7491 A) and the like. It is done.
[0035]
The ORF1554 used in the present invention encodes an ORF1554 product containing one or several amino acid substitutions, deletions, insertions or additions at one or more positions, so long as the ORF1554 product activity of the encoded protein is not impaired. You may do. Here, “several” differs depending on the position and type of the protein in the three-dimensional structure of amino acid residues, but specifically 2 to 30, preferably 2 to 20, and more preferably 2 to 10 It is a piece.
Specific examples of the ORF1554 product having an amino acid substitution as described above include, for example, an ORF1554 product having an amino acid sequence in which the glutamic acid residue at position 81 in SEQ ID NO: 2 is substituted with a glycine residue.
[0036]
DNA encoding a protein that is substantially identical to the ORF1554 product as described above may contain a substitution, deletion, insertion, addition, or inversion of an amino acid residue at a specific site, for example, by site-directed mutagenesis. Further, it can be obtained by modifying the base sequence of ORF1554. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. As the mutation treatment, a method of treating DNA before mutation treatment with hydroxylamine or the like in vitro, and a microorganism that retains the DNA before mutation treatment, such as an Escherichia bacterium, are irradiated with ultraviolet rays or N-methyl-N′-nitro-N -A method of treating with a mutating agent usually used for mutating treatment such as nitrosoguanidine (NTG) or nitrous acid.
[0037]
By expressing the DNA having the mutation as described above in an appropriate cell and examining the activity of the expression product, DNA encoding a protein substantially identical to the ORF1554 product can be obtained. Further, from a DNA encoding the ORF1554 product having a mutation or a cell holding the same, for example, a probe having a base sequence consisting of base numbers 749 to 1414 or a part thereof among the base sequences described in SEQ ID NO: 1 of the sequence listing; DNA that hybridizes under stringent conditions and encodes a protein having ORF1554 product activity is obtained. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, highly homologous DNAs, for example, 50% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90%. 60 ° C., 1 × SSC, 0.1%, which is a condition in which DNAs having the above homology hybridize and DNAs having lower homology do not hybridize, or washing conditions of normal Southern hybridization Conditions for hybridizing at a salt concentration corresponding to SDS, preferably 0.1 × SSC, 0.1% SDS are mentioned.
[0038]
A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the base sequence of SEQ ID NO: 1 as a primer and a DNA fragment containing the base sequence of SEQ ID NO: 1 as a template. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.
[0039]
Specifically, DNA encoding a protein substantially identical to the ORF1554 product is preferably 50% or more, more preferably 70% or more, still more preferably 80%, particularly preferably the amino acid sequence shown in SEQ ID NO: 2. Examples include DNA encoding a protein having 90% or more homology and having ORF1554 product activity.
[0040]
In addition, the coryneform bacterium of the present invention may have an enhanced activity of an enzyme that catalyzes L-glutamic acid biosynthesis, in addition to being modified to increase the activity of the ORF1554 product. Enzymes that catalyze biosynthesis of L-glutamate include glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconite hydratase, citrate synthase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase Phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, furtose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and the like.
[0041]
Furthermore, the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid may be reduced or deficient. As an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid, α-ketoglutarate dehydrogenase (αKGDH), isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetokinase Examples include hydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like. Of these enzymes, αKGDH is preferred.
[0042]
The αKGDH is encoded by the sucA gene. Coryneform bacteria in which the sucA gene is disrupted are described in detail in WO95 / 34672 International Publication Pamphlet and JP-A-7-834672.
In addition, since a sucA gene disruption strain may worsen growth compared with a parent strain, it is preferable to select a strain with good growth using an appropriate medium. Examples of the medium include CM2B plates (10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 10 μg / L biotin, 20 g / L agar, pH 7.0).
[0043]
<2> Method for producing L-glutamic acid
L-glutamine can be efficiently produced by culturing a coryneform bacterium obtained as described above in a medium, producing and accumulating L-glutamine in the medium, and collecting L-glutamine from the medium. it can.
[0044]
In order to produce L-glutamine using the coryneform bacterium of the present invention, a normal medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary is usually used. Can be done by law. Either synthetic or natural media can be used. Any kind of carbon source and nitrogen source may be used as long as the strain to be cultured can be used.
[0045]
As the carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses are used, and other organic acids such as acetic acid and citric acid, and alcohols such as ethanol alone. Alternatively, it is used in combination with other carbon sources.
[0046]
As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates, and the like are used.
[0047]
Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soybean protein breakdown products, etc. containing these, and auxotrophic mutations that require amino acids for growth. When using a strain, it is preferable to supplement the required nutrients.
[0048]
As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
The culture is carried out with aeration while controlling the fermentation temperature at 20 to 45 ° C. and the pH at 5 to 9. If the pH drops during the culture, add calcium carbonate or neutralize with an alkali such as ammonia gas. Thus, by culturing for about 10 to 120 hours, a significant amount of L-glutamine is accumulated in the culture solution.
[0049]
The method for collecting L-glutamine from the culture solution after completion of the culture may be performed according to a known recovery method. For example, the cells are collected by removing concentrated cells from the culture and then concentrating the crystals.
[0050]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0051]
[Example 1] Acquisition of acid-resistant mutant from wild Brevibacterium lactofermentum
Brevibacterium lactofermentum wild strain ATCC13869 was mutagenized using the mutagen N-methyl-N′-nitro-N-nitrosoguanidine (NTG) by the following method. The ATCC13869 strain was absorbed in two 4 ml test tubes containing 8 ml of CM2B medium (10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 10 μg / L biotin, pH 7.0) at 660 nm (OD ₆₆₀ ) Was shaken until about 0.7. The cultured cells were washed twice with 50 mM phosphate buffer (pH 7.0) and suspended in 500 μl of 50 mM phosphate buffer (pH 7.0). To this cell suspension, add NTG to a final concentration of 0.5 μg / μl, incubate at 31.5 ° C. for 10 minutes, wash with 3 × 50 mM phosphate buffer (pH 7.0), 200 μl of 50 mM It was suspended in a phosphate buffer (pH 7.0). The cells were inoculated into 8 ml CM2B medium and cultured overnight at 31.5 ° C. to prepare mutant-treated cells.
[0052]
In order to concentrate acid-resistant mutants from the above-mentioned mutant-treated cells, continuous culture under acidic conditions was performed using an S-type jar fermenter. The method is as follows. 300ml medium (60g / L glucose, 1.4g / LH _Three PO _Four 750mg / L MgSO _Four ・ 7H ₂ O, 15mg / L FeSO _Four ・ 7H ₂ O, 1.35g / L (as N) Soybean hydrolyzate ("Mameno" (Ajinomoto Co., Inc.), 450μg / L Vitamin B ₁ -HCl, 450μg / L biotin, 3μg / L vitamin B ₁₂ , 7.5 mg / L PABA (paraaminobenzoic acid), 7.5 mg / L vitamin C, 500 mg / L DL-methionine, 1 ml / L antifoaming agent AZ-20R (manufactured by NOF Corporation)) And inoculated at 31.5 ° C. After glucose is completely consumed, feed solution (same medium as the above medium) is added, and the culture solution is withdrawn simultaneously with the feed so that the amount of the medium is kept constant at 450 ml. Continuous culture was performed. The culture was started at pH 7.0 and cultured for 6 days. The pH of the medium gradually decreased with the passage of the culture time and was cultured until it reached pH 4.9.
[0053]
Screening of acid-resistant mutant strains was performed from the above-mentioned continuously cultured cells. The screening method is as follows. Continuously cultured microbial cells were adjusted to pH 5.7 (adjusted with KOH) MM / MES medium (20 g / L glucose, 10 g / L (NH _Four ) ₂ SO _Four , 1g / L KH ₂ PO _Four , 0.4g / L MgSO _Four ・ 7H ₂ O, 10mg / L FeSO _Four ・ 7H ₂ O, 10mg / L MnSO _Four ・ 4-5H ₂ O, 200μg / L vitamin B ₁ -HCl, 50 μg / L biotin, 5 mg / L nicotinamide, 1 g / L NaCl, 1 g / L casamino acid, 50 mg / L L-tryptophan, 30 mg / L L-cysteine, 100 mM MES (2- (N-morpholino) ethane Sulfonic acid), 20g / L agar), about 10 per plate ^Four The cells were uniformly spread so as to have a cell density of individual cells, and colonies formed after 3 days of incubation at 31.5 ° C. were obtained. Since wild-type strains cannot form colonies at pH 5.7, the strain obtained by this operation is considered to be an acid-resistant mutant strain that can form colonies under acidic conditions. Thus, acid-resistant mutant strains 16-1 and 16-20 were obtained. Further, acid-resistant mutant strains 15-11 were obtained by the same method except that a pH 5.9 medium was used and culture was performed for 3 days.
[0054]
[Example 2] Isolation of genes unique to Brevibacterium lactofermentum strains 16-1, 16-20, and 15-11
<1> Preparation of genomic library
Chromosomal DNA was prepared from each of the strains Brevibacterium lactofermentum 16-1, 16-20, and 15-11 obtained as described above, partially digested with the restriction enzyme Sau3AI, 4-6 kbp The DNA fragment was purified. These DNA fragments were inserted into the BamHI site of a plasmid vector (pSFK6) capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria. A genomic library consisting of about 14000 clones for 16-1 strain, about 7000 clones for 16-20 strain, and about 14000 clones for 15-11 strain was obtained.
[0055]
The pSFK6 is a plasmid prepared from the vector pHSG399 for Escherichia coli (see S. Takeshita et al: Gene 61, 63-74 (1987), which can be purchased from Takara Shuzo Co., Ltd.) and the kanamycin resistance gene of Streptococcus faecalis. This is a plasmid constructed from pK1 and plasmid pAM330 extracted from Brevibacterium lactofermentum ATCC 13869 (see U.S. Pat. No. 4,427,773, JP-A-58-67699) (JP-A-2000-262288, USA) Patent No. 6,303,383).
[0056]
<2> Acquisition of genes specific to acid-resistant mutants
The genomic libraries of 16-1, 16-20, and 15-11 strains were introduced into Brevibacterium lactofermentum ATCC 13869. An electric pulse method (Japanese Patent Laid-Open No. 2-207791) was used for introduction of the plasmid. The transformant was plated on an MM / MES plate having a pH of 5.7, and after culturing at 31.5 ° C. for 7 to 9 days, a clone forming a colony was obtained. Several hundred thousand clones were searched for each mutant genomic library. Brevibacterium lactofermentum ATCC13869 cannot form colonies on MM / MES plates with a pH of 5.7 or less, so the obtained clone is considered to have been given acid resistance by the gene on the plasmid. . Four types of such clones (clone # D5, clone # F1, clone # F2, and clone # H87) were obtained.
[0057]
Part of the base sequence of the genomic DNA fragment on the plasmid possessed by each of the clones # D5, # F1, # F2, and # H87 is shown in SEQ ID NOs: 1, 3, 5, and 7, respectively. In these sequences, open reading frames (ORF) (ORF1554: base numbers 749 to 1414 of SEQ ID NO: 1, ORF1249: base numbers 250 to 2748 of SEQ ID NO: 3, ORF39: base numbers 55 to 1212 of SEQ ID NO: 5, respectively. , ORF1059: base numbers 595 to 1644 of sequence 7). The amino acid sequences encoded by each ORF are shown in SEQ ID NOs: 2, 4, 6, and 8. The base numbers 1449 to 1455 of SEQ ID NO: 1, the base numbers 3317 to 3326 of SEQ ID NO: 3, and the base numbers 1 to 7 of SEQ ID NO: 5 are sequences derived from pHSG399.
[0058]
When the sequences of SEQ ID NOs: 1, 3, 5 and 7 were compared with the corresponding sequences on the genome sequence of Brevibacterium lactofermentum ATCC13869, the ORF1249, ORF39, and ORF1059 have unique mutation points in acid-resistant mutants. Although not observed, ORF1554 had a mutation point peculiar to acid-resistant mutant 16-1. In the mutant ORF1554 shown in SEQ ID NO: 1, the base at position 990 is “A”, whereas in the wild type, it is “G”. As a result, in the predicted amino acid sequence of the mutant ORF1554 product, position 81 of SEQ ID NO: 2 is Glu, whereas in the wild type, it is Gly. In order to distinguish between them, the wild type is ORF1554 and the mutant type is ORF1554. ^* May be written.
[0059]
<3> Introduction of ORF1554, ORF1249, ORF39, ORF1059 into wild Brevibacterium lactofermentum
XbaI of plasmid vector pSAC4 capable of autonomously replicating a DNA fragment having the base sequence shown in SEQ ID NO: 1 (having an XbaI recognition sequence at both ends) in both Escherichia coli and Corynebacterium bacteria The plasmid pD5-2A was prepared by inserting into the site. PSAC4 is obtained by digesting plasmid pHM1519 (Miwa, k. Et al., Agric. Biol. Chem., 48 (1984) 2901-2903) capable of autonomous replication in coryneform bacteria with restriction enzymes BamHI and KpnI. A plasmid obtained by blunt-ending the replication origin (JP-A-5-7491) and then inserting it into the SalI site of the Escherichia coli vector pHSG399 using a SalI linker (Takara Shuzo Co., Ltd.) It is.
[0060]
A DNA fragment having the base sequence shown in SEQ ID NO: 2 (having a BglII recognition sequence at the 3 ′ end and a KpnI recognition sequence at the 5 ′ end) was ligated to the BamHI site and KpnI site of pSAC4, and plasmid pF1-1B was Produced.
[0061]
A DNA fragment having the base sequence shown in SEQ ID NO: 3 (having an XbaI recognition sequence at both ends) was inserted into the XbaI site of pSAC4 to prepare plasmid pF2-2A.
A DNA fragment having the base sequence shown in SEQ ID NO: 4 (having a NaeI recognition sequence at both ends) was inserted into the SmaI site of pSAC4 to prepare plasmid pH87-4A.
[0062]
Each of the above four plasmids was introduced into Brevibacterium lactofermentum ATCC 13869. Each transformant was cultured on an MM / MES plate adjusted to an acidic pH at 31.5 ° C. for 5 days to examine colony formation. The results are shown in Table 1. As a result, in the ATCC13869 / pSAC4 strain (control strain) in which pSAC4 was introduced into Brevibacterium lactofermentum ATCC13869, colony formation was impossible at pH 5.7, but the plasmids pD5-2A, pF1-1B, and pF2-2A were not In the introduced ATCC13869 / pD5-2A strain, ATCC13869 / pF1-1B strain, and ATCC13869 / pF2-2A strain, colony formation was possible. At pH 6.0, larger colonies were formed with ATCC13869 / pH87-4A than ATCC13869 / pSAC4 strain (control strain). Thus, it was found that acid resistance can be imparted by introducing ORF1554, ORF1249, ORF39, and ORF1059.
[0063]
In addition, when plasmid pD5WT-1 (described later) carrying wild-type ORF1554 is introduced into ATCC13869, ORF1554 ^* (Table 1).
[0064]
[Table 1]

[0065]
<4> Preparation of plasmid carrying wild type ORF1554
The primers shown in SEQ ID NOs: 9 and 10 were designed with reference to the nucleotide sequence of SEQ ID NO: 1, and a region containing about 750 bp upstream and about 30 bp downstream of wild type ORF1554 from Brevibacterium lactofermentum ATCC 13869 was obtained by PCR. I got it. PCR was carried out using pyrobest DNA polymerase (Takara Shuzo Co., Ltd.), followed by 30 cycles of 94 ° C. for 5 minutes, 98 ° C. for 5 seconds, 65 ° C. for 10 seconds, and 72 ° C. for 60 seconds. The obtained PCR product of about 1.5 kb was cleaved at the XbaI site designed on both primers and inserted into the XbaI site of the plasmid vector pSAC4 to prepare pD5WT-1.
[0066]
<5> ORF1554, ORF1554 ^* L-glutamic acid production by gene amplification strain
ORF1554, ORF1554 ^* In order to examine the effect of gene amplification on L-glutamic acid production, plasmids pD5WT-1 (ORF1554) and pD5-2A (ORF1554) ^* ) Was introduced into the sucA gene-deficient strain L30-2 derived from Brevibacterium lactofermentum ATCC13869, and the amplified strain L30-2 / pD5WT-1 of ORF1554 and ORF1554 ^* An amplified strain L30-2 / pD5-2A was prepared. The plasmid was introduced by the electric pulse method (Japanese Patent Laid-Open No. 2-207791). The L30-2 strain was derived from the sucA gene-deficient strain ΔS strain (WO95 / 34672 international publication pamphlet) of Brevibacterium lactofermentum ATCC13869, CM2B plate (10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 10 μg / L biotin, 20 g / L agar, pH 7.0).
[0067]
Acquired ORF1554, ORF1554 ^* Amplified strains in CMDX plates (5 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 1 g / L KH ₂ PO _Four , 0.4g / L MgSO _Four ・ 7H ₂ O, 10mg / L FeSO _Four ・ 7H ₂ O, 10mg / L MnSO _Four ・ 4-5H ₂ O, 3 g / L urea, 2 g / L (as N amount) “Mino”, 20 g / L agar, 5 μg / L chloramphenicol, pH 7.5) at 30 ° C. overnight, 1/6 plate 20 ml of medium (30 g / L glucose, 15 g / L (NH _Four ) ₂ SO _Four , 0.4g / L MgSO _Four ・ 7H ₂ O, 1mg / L FeSO _Four ・ 7H ₂ O, 1mg / L MnSO _Four ・ 4-5H ₂ O, 200μg / L vitamin B1, 200μg / L biotin, 0.48g / L (as N amount) “Mino”, 1g / flask CaCO _Three Inoculate a flask containing 5 μg / L chloramphenicol, pH 8.0), shake culture at 30 ° C. for 20 hours, and then use biotech analyzer (Sakura Seiki) The concentration of L-glutamic acid was measured. FIG. 1 shows the yield of L-glutamic acid and the final OD (measured by diluting the culture solution 51 times, 620 nm) based on the consumed glucose. ORF1554 and ORF1554 ^* Both of these amplifications were found to increase the glutamic acid yield of Brevibacterium lactofermentum by about 4%.
[0068]
【The invention's effect】
According to the present invention, the ability to produce L-glutamic acid of Brevibacterium lactofermentum can be improved.
[0069]
[Sequence Listing]

[Brief description of the drawings]
[Figure 1] ORF1554 and ORF1554 ^* The growth and the L-glutamic acid yield of Brevibacterium lactofermentum sucA deficient strain which amplified A.

Claims (6)

In the method for producing L-glutamic acid, a coryneform bacterium having L-glutamic acid-producing ability is cultured in a medium, L-glutamic acid is produced and accumulated in the culture, and L-glutamic acid is collected from the culture. The method is modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) an activity comprising the amino acid sequence of SEQ ID NO: 2 comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and improving the ability to produce L-glutamic acid of coryneform bacteria A protein having The method according to claim 1, wherein the protein is encoded by DNA shown in the following (a) or (b).
(A) DNA having a base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1.
(B) DNA encoding a protein that hybridizes with the base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1 under the stringent conditions and has an activity of improving the ability to produce L-glutamic acid of coryneform bacteria.   The bacterium increases the copy number of the gene encoding the protein shown in (A) or (B), or the expression of the gene encoding the protein shown in (A) or (B) in the bacterial cell is increased. The method according to claim 1 or 2, wherein the activity of the protein in the cell is increased by modifying the expression regulatory sequence of the gene so as to enhance.   The method according to any one of claims 1 to 3, wherein the protein has an amino acid sequence in which the glutamic acid residue at position 81 of SEQ ID NO: 2 is substituted with a glycine residue.   The method according to claim 1, wherein the bacterium is deficient in α-ketoglutarate dehydrogenase. A coryneform bacterium having L-glutamic acid-producing ability and modified so that the activity of the protein shown in (A) or (B) below is increased.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) an activity comprising the amino acid sequence of SEQ ID NO: 2 comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and improving the ability to produce L-glutamic acid of coryneform bacteria A protein having

Images